APPLuZD MICROBIOLOGY, Sept. 1975, p. 477-479Copyright © 1975 American Society for Microbiology

Vol. 30, No. 3Printed in U.S.A.

Vitamin B12 Production by a Methanol-Utilizing BacteriumTETSUO TORAYA, BUSABA YONGSMITH,' ATSUO TANAKA, AND SABURO FUKUI*

Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Yoshida,Sakyo-ku, Kyoto, Japan

Received for publication 28 April 1975

Vitamin B12 production by a newly isolated strain of a methanol-utilizingbacterium was studied. The maximal yield of the vitamin, 2.6 mg/liter ofmedium, was attained by optimization.

A pink-pigmented bacterial strain capable ofutilizing methanol as a sole source of carbonand energy was isolated from soil of the oil fieldin Niigata, Japan. This new isolate, strain FM-02T, was found to produce vitamin B12 signifi-cantly. It would be of much practical signifi-cance to produce this expensive and compli-cated vitamin from cheap and simple noncar-bohydrate substrates, such as methanol, sincevitamin B12 is at present produced exclusivelyby fermentation of carbohydrates using certainbacteria. Recently, formation of vitamin B12 bymethanol-utilizing bacteria has been reportedby us (6) and by Nishio et al. (3, 4). However,the vitamin B12 productivities reported by themwere too poor for industrial application (lessthan 0.3 mg/liter). Therefore, we attempted tooptimize vitamin B12 production using ournewly isolated strain of a methanol-utilizingbacterium and obtained a maximal yield of 2.6mg/liter of medium.The composition of the basal medium used for

the methanol utilizer, strain FM-02T, in batchculture experiments is identical to that de-scribed before (6): NH$2PO4, 2.0 g; KH2PO4,2.0 g; Na2HPO, 12H20, 3.0 g; MgSO4 7H20, 0.2g; CaCl2 2H20, 0.01 g; FeSO4 7H20, 0.005 g;MnSO4,nH20, 0.005 g; CoSOg7H20, 0.001 g;and carbon source, 10 ml (liquid substrate) orthe amount corresponding to 0.3 g-atom of car-bon in 1 liter of tap water (pH 7.0 to 7.2).Cultivation was carried out aerobically at 30 C.Vitamin B12 compounds (consisting of adenosyl-cobalamin and methylcobalamin) in culturedbroth were extracted as cyanocobalamin by boil-ing in 0.08 M acetate buffer (pH 5.5) containing0.01% KCN, and the amount was determinedmicrobiologically using Escherichia coli 215, avitamin B12-L-methionine auxotroph, as a testorganism (2).

1 Present address: Department of Microbiology, Facultyof Sciences and Art, Kasetsart University, Bangkok, Thai-land.

Strain FM-02T is gram negative, pink pig-mented, and rod shaped. The absorption spec-trum of the main pigment fraction from thebacterium, which was extracted with absoluteethanol, was quite similar to that from Pseu-domonas AM1 (5). Strain FM-02T was able toutilize 1,2-propanediol and lactate, as well asmethanol, as sole carbon sources, indicatingthat this organism is a facultative methylo-troph. Poor growth was observed on succinate,fumarate, malate, and tartrate. Glycerol, meth-ylamine, and dimethylamine also permittedsome growth after a long lag period. Among thecarbon sources tested, the following compoundsdid not appreciably serve as growth substrates:formate, trimethylamine, ethanol, ethylene gly-col, acetate, glycine, n-propanol, propionate,serine, glutamate, citrate, and glucose.

Effects of growth conditions on vitamin B12productivity were then studied. When culturedon a rotary shaker (200 rpm) at 30 C, thegrowth reached maximum in 3 days in thebasal medium (1% methanol [vol/voll), whereasthe amount of vitamin B12 produced reachedmaximum in 2 days (late exponential phase).Almost all of the vitamin was found to be in-cluded in the cells. Vitamin B12 productivitywas dependent on Co2+ concentration, and 1 mgof CoSO4 7H20 per liter was used as the optimalconcentration for usual experiments. StrainFM-02T grew most rapidly in the medium con-taining methanol at an initial concentration of0.8 to 1.2% (Fig. 1). The growth was markedlyinhibited by a higher concentration of metha-nol, and essentially no growth was observed on4.8% methanol. It seems interesting that maxi-mal production of the vitamin was obtained at2.4%, where the bacterial growth was inhibitedto some extent. Effects of addition of variousnutrients on vitamin B12 productivity were ex-amined (Table 1). Among the natural nutrientstested, Casamino Acids (Difco) enhanced vi-tamin B12 production significantly. L-Methio-

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1.0

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1 2 3 4INITIAL METHANOL CONCENTRATION (%,v/v)

FIG. 1. Effects of initial concentration of methanol on growth and vitamin B12 production. The bacteriumwas cultivated for 3 days with shaking.

TABLE 1. Effects of various nutrients on vitamin B12production

Growth Vitamin B12(g of produced

Expt Nutrienta dry

liter) liter cells

1 None 2.4 110 46Casamino Acids 2.4 153 64Corn steep liquor 2.4 120 50Malt extract 2.4 116 48Meat extract 2.4 96 40Peptone 1.9 85 45Yeast extract 2.2 110 50

2 None 2.2 109 50Glycine 2.4 72 30L-Serine 2.3 109 47L-Aspartate 1.9 126 66L-Glutamate 1.9 80 42L-Methionine 1.9 140 74L-Threonine 0.5 14 28

a Natural nutrient, 0.1%; amino acid, 1 mM. Thebacterium was cultivated for 3 days with shaking.

nine also stimulated the vitamin production.The following compounds did not affect the vi-tamin productivity: glycine and/or succinate(precursors of corrin ring); adenosine, adenine,

and related compounds (precursors of adenosylgroup of coenzyme B12); betaine or choline(methyl donor); and various vitamins. Penicil-lin G and various surfactants also did not affectvitamin B12 production.The yield of the vitamin was not as high even

after optimization of the growth conditions inthe shaking culture. Therefore, the cultivationmethod was improved to obtain the cells inmuch larger quantities. "Exponential-fed batchcultivation" was found to be significantly favor-able for bacterial growth as well as vitamin B12formation. In this cultivation method, the feedrate of methanol was increased exponentiallyin accord with the exponential growth of themicroorganism by use of a rotating drum-typeprogrammer, thus keeping the methanol con-

centration in the culture medium at a constantlow level. (Details of this method will be pub-lished elsewhere by F. Yoshida and his co-work-ers). By using this method, about 20 g of drycells and 0.4 mg of vitamin B12 were obtainedper liter of the medium (Table 2). As far as

bacterial growth is concerned, it would be pref-erable to keep the methanol concentration as

low as possible. However, the higher methanolconcentration was more suitable for vitaminB12 production under our experimental condi-tions. The yield of vitamin B12 further in-creased to about 2.6 mg/liter of medium by

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478 NOTES

TABLE 2. Optimization of vitamin B12 production

L-Methio- Casamino Cultiva- Vitamin B12 produced-Mto-Casamino Cutia Growth _________Cultivation method Methanol concn nine Acids tion (g of dry(%, vol/vol) (1 mM) (0.1%) time cells/liter) liter dLglgof

Batcha 1 (initial) - - 72 2.2 108 491 (initial) + + 72 1.7 130 762.4 (initial) - - 72 2.3 116 502.4 (initial) + + 72 2.7 172 64

Exponential-fed 0-0.05 - - 26.0 19.8 400 20batchb 0-0.95 - - 31.3 17.2 863 50

0.40-1.38 +- 48.2 25.0 2,560 102

a The bacterium was cultivated in a 500-ml shaking flask containing 100 ml of the medium (initial pH, 7.0to 7.2) at 30 C on a rotary shaker (200 rpm).

b The bacterium was cultivated in a 10-liter jar fermenter (working volume, 5 liters) at 30 C with agitationspeed of 300 to 1,500 rpm and aeration rate of 2 or 10 liters/min.

c Methionine (3.3 mM) was added at a cell concentration of 10 g of dry cells per liter (at 25.8 h).

increasing the methanol concentration and add-ing L-methionine. Although the value is stilllower than the highest values obtained by in-dustrial-type microorganisms cultivated on car-bohydrate media (23 mg/liter byPropionibacter-ium shermanii; 5.7 mg/liter by Streptomycessp.) (7), the use of methanol for microbial pro-duction of vitamin B12 is promising and attrac-tive from a practical point of view.Paper electrophoretic behaviors of the B12

fraction from strain FM-02T demonstrated thatthe vitamin exists mainly in the forms of adeno-sylcobalamin (coenzyme B12) and methylcobala-min. The coenzyme activity of the fraction inthe coenzyme B12-dependent diol dehydrase(D,L-1,2-propanediol hydro-lyase, EC 4.2.1.28)system (1) from Aerobacter aerogenes ATCC8724 gave additional clear evidence for the pres-ence of adenosylcobalamin in the cells of strainFM-02T (data not shown).

We are grateful to Fumitake Yoshida, Tsuneo Yamane,and Michimasa Kishimoto, Department of Chemical Engi-

neering, Kyoto University, for cultivating the cells usingthe "exponential-fed batch technique." Thanks are also dueto Susumu Honda for the microbiological assay of vitaminB12.

LITERATURE CITED1. Abeles, R. H., and H. A. Lee, Jr. 1961. An intramolecu-

lar oxidation reduction requiring a vitamin B12 coen-zyme. J. Biol. Chem. 236:PC1.

2. Ikeda, H. 1956. The microbiological assay of vitamin B12utilizing auxotrophic mutants ofEscherichia coli (I). Abasic study. Vitamins (Kyoto) 10:268-279.

3. Nishio, N., T. Yano, and T. Kamikubo. 1975. Isolation ofmethanol-utilizing bacteria and its vitamin B12 produc-tion. Agric. Biol. Chem. 39:21-27.

4. Nishio, N., T. Yano, and T. Kamikubo. 1975. Furtherstudies ofvitamin B12 production by methanol utilizingbacterium, Klebsiella sp. no. 101. Agric. Biol. Chem.39:207-213.

5. Peel, D., and J. R. Quayle. 1961. Microbial growth on C,compounds. 1. Isolation and characterization of Pseu-domonas AM1. Biochem. J. 81:465-469.

6. Tanaka, A., Y. Ohya, S. Shimizu, and S. Fukui. 1974.Production of vitamin B12 by methanol-assimilatingbacteria. J. Ferment. Technol. 52:921-924.

7. Wuest, H. M., and D. Perlman. 1968. Vitamin B12. III.Industrial preparation and production, p. 139-144. InW. H. Sebrell, Jr., and R. S. Harris (ed.), The vita-mins, vol. II. Academic Press Inc., New York.

VOL. 30, 1975 NOTES 479